Inactivation of a GAL4 -Like Transcription Factor Improves Cell Fitness and Product Yield in Glycoengineered Pichia pastoris Strains

Abstract

bWith a completely reengineered and humanized glycosylation pathway, glycoengineered Pichia pastoris has emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineered Pichia strains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within the Pichia pastoris ATT1 (PpATT1) gene (a homolog of the Saccharomyces cerevisiae GAL4 [ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineered Pichia strains. We demonstrate that deletion of the PpATT1 gene enabled glycoengineered Pichia strains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled the PpATT1-modified glycoengineered Pichia strains to increase their product yields significantly without any sacrifice in product quality. Because the ATT1 gene could be deleted from any Pichia strains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to any Pichia strains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors. Since the approval of the first biopharmaceutical product (recombinant insulin) in 1982, biopharmaceuticals, as a prescription drug class, have enjoyed the highest growth rate within the pharmaceutical industry (1). With more than 230 approved products currently on the market, biopharmaceuticals are playing vital roles in the prevention and treatment of a wide variety of diseases, ranging across infectious diseases, inflammatory disorders, metabolic diseases, and cancer. Most biopharmaceuticals are manufactured from one of three different expression host systems: mammalian cells, yeasts, and bacteria (2). Bacterial systems (e.g., Escherichia coli) are the industry standard expression hosts for proteins that do not require posttranslational modifications (e.g., insulin, growth hormone, granulocyte colony-stimulating factor [G-CSF]). To date, mammalian cells (e.g., CHO) have been the preferred expression system for glycoproteins and monoclonal antibodies (MAbs), primarily due to their ability to produce glycosylation patterns adequate for drug safety and efficacy. Despite the advantages of being unicellular eukaryotes with efficient protein-folding and posttranslational-modification capabilities, yeasts are used for the production of only a small number of therapeutic proteins, because their high-mannose glycosylation patterns are not optimal for drug safety and efficacy.Glycoengineered Pichia pastoris has recently emerged as a promising production host for the manufacture of therapeutic ...

The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris

“…Previous studies also identified mutations of Gal4-like transcription factors and TUP1 to be associated improved growth of P. pastoris . [ 25 , 27 ]; therefore these regulatory hubs and the cAMP/Pka pathway could represent interesting engineering targets for growth optimization of recombinant P. pastoris host strains, specifically in minimal growth conditions.…”

“…Therefore OCH1 node removal, although leading to a viable phenotype, has already been previously reported to lead to abnormal growth and stress resistance in yeasts [ 21 , 24 ]. As a consequence, attempts have already been made to improve growth of glyco-engineered yeasts [ 25 ].…”

Transcriptional engineering of the glyceraldehyde‐3‐phosphate dehydrogenase promoter for improved heterologous protein production in Pichia pastoris

“…The highest expression level obtained by this variant is 1.5‐fold higher relative to wild‐type P GAP in limited glucose conditions. As discussed previously, there are more than 20 Gal4‐ like TFs encoded in the P. pastoris genome and these TFs appear to have distinct regulatory effects on cell physiology (Jiang et al, ; Moser et al, ). Therefore, it is likely that at least one Gal4 ‐like TF is responsible for regulation of P GAP as an activator, yet the details of the mechanism are still unknown.…”

“…Therefore it is likely that these Gal4 ‐like TFs are responsible for regulation of other pathways. Indeed, Jiang et al () referred to one of these Gal4 ‐like TFs (PP7435_Chr1‐1522) as Pp Att1 as it shows a significant sequence homology to Sc Gal4. They demonstrated that knocking‐out this TF improved thermal tolerance levels, reduced cell lysis defects, and improved robustness in bioreactor cultivation of a glycoengineered P. pastoris strain.…”

Recent Advances in Pichia pastoris as Host for Heterologous Expression System for Lipases: A Review

“…The co-expression of folding and secretion factors (9-10), the optimization of the properties of foreign nucleotide sequence (codon usage) (11), the insertion of multiple recombinant gene copies (12), engineering of glycosylation (13) and secretory pathways (14) continue as topics to improve the performance of Pichia as cell factory (16).…”